Program Abstract- Project 3 Encephalitic alphaviruses (i.e., Venezuelan, Western, and Eastern Equine Encephalitis viruses) can cause devastating encephalitis in humans and equids. Several drug discovery efforts have been pursued to fulfill the unmet needs for effective antivirals for these viruses; however, they are still not available for treatment. Here we propose the development of a therapeutic candidate for encephalitic alphaviruses based on our two novel lead series. Prototypes within each series showed potent antiviral activity in vitro and in vivo without adverse effects. In Aim 1, we will evaluate newly designed compounds in vitro for their potentials as anti-VEEV, EEEV therapeutics to support the lead optimization of Research Project 1 and contribute to the selection of leads to be tested in vivo in Research Project 2. In Aim 2, we will contribute to experimental studies and documentation for the Non-Clinical Virology Study Report for the selected lead candidate in preparation for the informal and Pre- IND meetings in collaboration with Leidos. We will experimentally establish that the alphaviral RNA replicase is the target of the lead candidate compounds, using in vitro viral RNA synthesis assays. Specifically, we will evaluate the anti-V/W/EEEV activity in biologically relevant systems, and importantly, measure the Inhibitory Quotient to demonstrate antiviral activity in human blood. We will also determine the in vitro safety and therapeutic index of the lead candidate compounds and define the resistance threshold of resistant viruses.